This invention relates to the isolation and purification of deoxyribonuclease from crude or partially purified preparations of the enzyme. Deoxyribonuclease enzymes, hereinafter termed "deoxyribonuclease" or "DNase", are widely distributed in nature and are present in substantial amounts in the pancreas, blood and spleen of animals as well as in yeasts. A good source of deoxyribonuclease is the bovine pancreas, however, porcine pancreas and bovine spleen may also serve as source materials.
Deoxyribonuclease has several medicinal and veterinary uses. One use is as a debriding agent to liquefy pus in order to aid in the removal of necrotic debris from skin surfaces.
Numerous methods are described in the literature for the isolation and purification of DNase from bovine pancreas. The enzyme is customarily extracted into dilute sulfuric acid, followed by a series of ammonium sulfate fractionations, heat treatment, alcohol fractionation and ammonium sulfate crystallization at acidic pH. The yield of crystalline DNase is in the order of 3-5 mg/kg pancreas, and it is often contaminated with large amounts of chymotrypsin B or its zymogen. A modified sulfuric acid extraction procedure, purported to give higher yields (2-3 times as much) of crude DNase/kg pancreas is disclosed in U.S. Pat. No. 2,801,956.
Other methods for obtaining highly purified deoxyribonuclease involve electrodecantation, chromatography on either cation exchangers or anion exchangers, and affinity chromatography on agarose-bound DNA. While the foregoing methods have been reported to yield highly pure deoxyribonuclease, the starting materials were either purified (70% pure) or crystalline enzyme. Furthermore, treatment of these starting materials with a highly toxic proteolytic enzyme inhibitor, such as diisopropylfluorophosphate (DFP) was always necessary to ensure a recovery of purified enzyme greater than 50%. Of the foregoing methods, affinity chromatography on a column of agarosebound DNA appears to be the most selective and simple. However, this method has the disadvantage of having a low column capacity when a crude enzyme source is used as a starting material in the purification process.